Ceragenin-mediated disruption of Pseudomonas aeruginosa biofilms

Background Microbial biofilms, as a hallmark of cystic fibrosis (CF) lung disease and other chronic infections, remain a desirable target for antimicrobial therapy. These biopolymer-based viscoelastic structures protect pathogenic organisms from immune responses and antibiotics. Consequently, treatments directed at disrupting biofilms represent a promising strategy for combating biofilm-associated infections. In CF patients, the viscoelasticity of biofilms is determined mainly by their polymicrobial nature and species-specific traits, such as Pseudomonas aeruginosa filamentous (Pf) bacteriophages. Therefore, we examined the impact of microbicidal ceragenins (CSAs) supported by mucolytic agents–DNase I and poly-aspartic acid (pASP), on the viability and viscoelasticity of mono- and bispecies biofilms formed by Pf-positive and Pf-negative P. aeruginosa strains co-cultured with Staphylococcus aureus or Candida albicans. Methods The in vitro antimicrobial activity of ceragenins against P. aeruginosa in mono- and dual-species cultures was assessed by determining minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). Inhibition of P. aeruginosa mono- and dual-species biofilms formation by ceragenins alone and in combination with DNase I or poly-aspartic acid (pASP) was estimated by the crystal violet assay. Additionally, the viability of the biofilms was measured by colony-forming unit (CFU) counting. Finally, the biofilms’ viscoelastic properties characterized by shear storage (G’) and loss moduli (G”), were analyzed with a rotational rheometer. Results Our results demonstrated that ceragenin CSA-13 inhibits biofilm formation and increases its fluidity regardless of the Pf-profile and species composition; however, the Pf-positive biofilms are characterized by elevated viscosity and elasticity parameters. Conclusion Due to its microbicidal and viscoelasticity-modifying properties, CSA-13 displays therapeutic potential in biofilm-associated infections, especially when combined with mucolytic agents.


Introduction
Biofilm formation is a colonization and survival strategy exploited by numerous microorganisms, such as Pseudomonas aeruginosa and other cystic fibrosis (CF) pathogens, often leading to chronic infections.Biophysically, these microbial communities embedded in a matrix of autogenic extracellular polymers (EPS) are characterized by viscoelastic, i.e., either solid (elastic) and liquid (viscous) qualities, that correlate with their resistance to mechanical stress as well as eradication by immune responses and antibiotics [1,2].Compared to their planktonic counterparts, bacteria living in biofilms can be up to a thousand times more resistant to antibiotics.Consequently, understanding biofilm viscoelastic properties is crucial for developing effective treatments for biofilm-associated infections.
The remarkable adaptability of biofilms is due to the varied assemblage of species and their EPS matrix components.The latter account for 90% of biofilm biomass, primarily consisting of polysaccharides, extracellular DNA (eDNA) and RNA (eRNA), proteins, and lipids, which collectively are termed the 'matrix' [3,4] Also, host biomolecules, e.g., soluble proteins, glycoproteins, polymeric fibrin, F-actin and DNA, can be incorporated into the matrix, hence its composition and structure adapt to the local environment, availability of nutrients/substrates as well as local shear stress [3,5].For instance, highly viscoelastic CF mucus, which is osmotically hyperconcentrated and rich in DNA and mucin, and associated with low mucociliary transport in CF lungs [6,7], promotes robust biofilm formation by P. aeruginosa and other CF pathogens, including S. aureus, Haemophilus influenzae, Burkholderia cepacia complex (BCC), and C. albicans [8][9][10][11].The polymicrobial nature of CF biofilms substantially increases matrix heterogeneity, which along with species-specific factors, such as various exopolysaccharides (Psl-polysaccharide synthesis locus, Pel-pellicle polysaccharide, and alginate) produced by P. aeruginosa, strongly determine their viscoelasticity [12].
In response, various biopolymer-disrupting mucolytic treatments have been developed, such as the degradation of DNA by DNases and disruption of DNA/F-actin by anionic polyaspartic acid (pASP) [13][14][15][16].Similarly, targeting P. aeruginosa filamentous bacteriophages (Pf), carried as prophages by multiple clinical isolates, e.g., Liverpool epidemic strain (LES), may provide an alternative therapeutic approach for CF patients [17].The presence of Pf phages in sputum was correlated with a higher risk of chronic infection, a more significant decline in pulmonary function during exacerbations, and the development of antibiotic resistance compared to Pf-negative sputum samples [18].Likewise, Pf-producing P. aeruginosa strains were associated with delayed healing in patients with chronic wound infections [19], and modulation of immune response toward impairing bacterial clearance at the site of infection [20].Furthermore, Pf phages exist in two evolutionary lineages (I and II) with significantly different properties, such as the net charge of the capsid protein, which may affect interactions with other matrix biomolecules [21].
As a result, effective treatment of biofilm-associated infections necessitates a combination of agents disrupting structural components of the matrix and killing microbial cells, preferably by antimicrobials that maintain activity in this biomolecule-rich niche.Ceragenins are nonpeptide mimics of endogenous antimicrobial peptides (AMPs), developed to overcome the therapeutic limitations of AMPs, e.g., inactivation by anionic biopolymers, high salt concentrations, or proteases [22,23].Ceragenins are less susceptible to inactivation by DNA or F-actin compared to AMPs, and their antibacterial action is not impeded by Pf phages [24].Additionally, ceragenins possess a broad-spectrum microbicidal activity, low resistance potential, and show no cross-resistance to other antimicrobials.Moreover, they act synergistically with conventional antibiotics against multiple bacterial strains, including multidrug-resistant (MDR) ones [25][26][27], and possess anti-inflammatory properties [28].Hence, ceragenins can potentially eradicate biofilms in CF airways [28,29].
To test the ability of ceragenins to disrupt biofilms, we evaluated their antibacterial activity in combination with the viscoelasticity-reducing agents, DNase I and poly-aspartic acid (pASP), against mono-and bi-species biofilms produced by P. aeruginosa Pf-positive and Pfnegative strains with either S. aureus or C. albicans.We show that the treated biofilms are more vulnerable to the microbicidal action of ceragenins, likely due to reduced viscoelasticity, regardless of the species composition and Pf-patterns of P. aeruginosa strains.

Microbial strains
The study involved clinical strains of P. aeruginosa (n = 10) isolated from sputum samples collected from CF patients at the Adult Cystic Fibrosis Center at the University of Pennsylvania Health System (recruitment of participants were between 8/5/2005-5/23/2008) in the United States.Sputum samples and bacterial isolates were obtained with the agreement of The University of Pennsylvania's Institutional Review Board (IRB) (no.803255) in compliance with the Helsinki Declaration and written informed consent was obtained from each patient.
The antibiotic susceptibility of P. aeruginosa isolates was determined using the VITEK 1 2 Compact system and Gram-negative Susceptibility Cards AST-N331 (bioMe ´rieux, Marcyl'Etoile, France), and interpreted using EUCAST ver.11 guidelines.Staphylococcus aureus Xen30 (Caliper Life Sciences, Hopkinton, MA, USA) and Candida albicans 1408 (Polish Collection of Microorganisms, Polish Academy of Science, Wroclaw, Poland) were used to develop dual-species biofilm models.

Determination of CSAs activity against the planktonic growth of P. aeruginosa in mono-and dual-species cultures
The minimum inhibitory concentrations (MIC) for CSA-13, CSA-44, and CSA-131 were determined in 96-well microtiter plates using two-fold serial dilutions of the ceragenins, ranging from 0.25 to 256 μg/mL, in Mueller Hinton (MH) and a final P. aeruginosa density of 5 × 10 5 cfu/mL.To do so, bacteria at the logarithmic phase of growth were collected and adjusted in LB broth to the OD ~0.1.The plates were incubated at 37˚C for 24 hours, followed by visual inspection for the MIC values, i.e., concentrations with no observable bacterial growth.In addition, two randomly selected Pf-positive and Pf-negative P. aeruginosa strains were mixed with S. aureus X30 or C. albicans 1408 cells to estimate the antibacterial effects of ceragenins against dual-species cultures using the same procedure.

Determination of CSAs activity against P. aeruginosa mono-and dualspecies biofilms
To estimate the ability of CSA-13 to suppress P. aeruginosa biofilm formation, bacterial strains at a final inoculum of 10 5 CFU/mL were incubated in MH broth with varied doses (5 and 20 μg/mL) of the ceragenins in 96-well microtiter plates to a final volume of 200 μL for 48h at 37˚C.After incubation, planktonic cells were removed, and the attached cells were washed three times with PBS.As evidenced in earlier reports, 48-hour incubation in high nutrient medium is sufficient to form a mature biofilm by Pseudomonas isolates [31,32].The biofilm was stained with 0.1% (w/v) crystal violet.After 15 minutes, the crystal violet solution was removed, and the plates were carefully washed with PBS for 2 seconds.To determine the mass of the biofilm, the remaining biofilm-staining crystal violet was dissolved in 95% ethanol.Biofilm intensity in the 96-well plated were measured at 570 nm using a Labsystem Varioscan Lux (Thermo Fisher Scientific, Waltham, MA, USA).The 50% inhibitory concentrations (IC 50 ) were calculated based on percentage inhibition with the different concentrations of CSAs compared with the non-treated biofilm.
In corresponding experiments, the mass of P. aeruginosa biofilms was evaluated in the presence of DNase I at concentrations ranging from 0.5 to 5 μg/mL, pASP at concentrations ranging from 1 to 200 μg/mL, and Pf1 bacteriophage at concentrations ranging from 0.01 to 0.5 mg/mL, that were administered alone and in combination with CSA-13 at doses of 5 or 20 μg/mL.
Likewise, the same procedure was applied for polymicrobial biofilms formed by Pf-positive and Pf-negative P. aeruginosa strains co-cultured with S. aureus and C. albicans.Recorded values were compared to the anti-biofilm activity of CSA-13 alone.

Estimation of biofilm viability
A colony-forming units (CFUs) counting technique was used as a complementary method to determine the microbicidal action of the compounds in P. aeruginosa dual-species biofilms.Briefly, the viability of the biofilms formed by Pf-positive and Pf-negative P. aeruginosa strains with S. aureus, and C. albicans strains was examined in the presence of with CSA-13 at a dose of 20 μg/mL alone and when co-administrated with DNase I, pASP as well as exogenous Pf1 phage at concentrations of 5 μg/mL, 10 μg/mL, and 0.1 mg/mL, respectively.After 48 hours of incubation, biofilms were sonicated for 5 minutes, diluted, and plated on tryptic soy agar plates supplemented with 5% sheep blood.After incubating the plates for at least 24 hours, the number of CFUs per biofilm sample was calculated.

Evaluation of rheological properties of biofilms
Rheological measurements were performed on a HAAKE Rheostress 6000 rotational rheometer (Thermo Scientific) using a Peltier system to maintain a constant temperature of 21˚C.Untreated and treated 10-day-old mono and dual-species biofilms were collected in plastic tubes and applied to the bottom plate of the rheometer.In the measurements of the viscoelastic properties, the forced oscillation method in the plate-plate arrangement (with a plate diameter of 35 mm) was used.In short, the upper plate rotates by a specified angle (shear strain amplitude γ 0 ) and frequency (ω), applying oscillatory shear strain to the sample, according to the equation: γ(ω,t) = γ 0 sin(ωt).The stress measured in this way can be described by the equation: τ(ω,t) = G'γ 0 sin(ωt) + G"γ 0 cos(ωt), where G' is storage shear strain modulus and G" is loss shear strain modulus.The research protocol was as follows: (1) amplitude sweep tests carried out in the range of 0.01-100 with a fixed frequency of 1 Hz, and (2) frequency sweep in the range of 0.1-10 Hz with a fixed amplitude of 0.01 (range of linear viscoelasticity).In addition, viscosity measurements were performed in rotational tests in the cone (diameter 35 mm, angle 1 degree)-plate arrangement, where the dynamic viscosity was determined in the range 0.1-100 1/s of shear rate (linearly related to the rotational cone velocity).

Statistical analysis
Data are presented and mean ± SE.The significance of differences was determined using the two-tailed Student's t-test.Statistical analyses were performed using OriginPro 2021 (Origi-nLab Corporation, Northampton, USA).p <0.05 was considered statistically significant.

Filamentous phages (Pf) profiles of P. aeruginosa strains
All Pf-positive P. aeruginosa isolates possessed unique Pf-profiles, consisting of one to four Pf phages belonging to four groups according to chromosome integration site, i.e., Pf-tRNA-Gly, Pf-tRNA-Met, Pf-tRNA-DR, Pf-tmRNA.The Pf-profiles and phyloproteomic relatedness of P. aeruginosa isolates are shown in S1 Fig.

Ceragenins inhibit the formation of P. aeruginosa biofilms
As shown in Fig 1B, there was no statistically significant variability between Pf-positive and Pfnegative P. aeruginosa strains in the 50%-level inhibitory concentration of ceragenins required to inhibit biofilm formation.CSA-13 exerted the most pronounced anti-biofilm action, with 50%-level inhibitory concentration at 11.35 μg/mL ± 3.91 and 9.71 μg/mL ± 1.22 for Pf-positive and Pf-negative P. aeruginosa groups, respectively.Based on this, ceragenin CSA-13 was selected for further experiments.
CSA-13 at a concentration of 5 μg/mL reduced the biofilm mass of Pf-negative isolates 13% ± 6% more than Pf-positive ones (Fig 1C).However, this effect was not confirmed by CSA-13 at a higher concentration-20 μg/mL, where the difference in biofilm masses was only 0.6% ± 0.9%.

The anti-biofilm action of CSA-13 is enhanced by DNase I and pASP
DNase I at concentrations of 0.5-5 μg/mL reduced the biofilm mass of Pf-negative and Pf-positive P. aeruginosa strains by 42-44% and 36-38%, respectively (Fig 2A).In contrast, pASP acid only slightly reduced biofilm formation in the Pf-negative group and appears to even promote biofilm formation in the Pf-positive group (Fig 2B).When a combination of DNase I or  acid as well as in biofilms supplemented with exogenous Pf1 phage was recorded (Fig 4A -4J).In general, the activity of the combination of CSA-13 with DNase I and pASP acid on dualbiofilms was similar to that exerted on the single-species biofilms, and considerable differences between biofilms formed by Pf-positive and Pf-negative isolates were noted only for selected concentrations of these compounds.For instance, CSA-13 only at a concentration of 20 μg/ mL significantly reduced the mass of P. aeruginosa Pf-negative dual-biofilms formed with S. .Biofilms of Pf-negative isolates show an increase in phase shift above strain 0.1 from ~15˚to over 45˚at strain 10 for Pf-negative + S. aureus (followed by a decrease to 30˚at strain 100) and from 30˚to over 60˚for Pf-negative P.

CSA-13 and DNase I decrease the stiffness and viscosity of biofilms
In the shear strain range of 0.01-1, a 4-fold decrease in storage modulus was observed for Pfnegative P. aeruginosa + S. aureus and nearly 2-fold for the other types (Fig 6B)  P. aeruginosa + C. albicans where a twofold decrease in viscosity was observed in the range up to 1 1/s.For Pf-negative P. aeruginosa + S. aureus and Pf-negative P. aeruginosa + C. albicans, the decrease in viscosity in this range was 2 and 1.5 times, respectively.At higher shear rates, viscosity was similar to untreated biofilms (S5G Fig) .The addition of DNase I also reduces viscosity, especially for biofilms with S. aureus, for which an almost 4-fold and 5-fold decrease in the storage modulus for Pf-negative and Pf-positive P. aeruginosa isolates, respectively (Fig 6C ) was observed.For P. aeruginosa with C. albicans, the storage modulus decreased by about 30% for Pf-negative P. aeruginosa and 45% with Pf-positive isolates (   for Pf-negative P. aeruginosa + C. albicans by over 80%, for Pf-positive P. aeruginosa + S. aureus by over 95% and for Pf-positive P. aeruginosa + C. albicans by over 90% (Fig 7A).At the same time, for all types of biofilms, a sharp decrease in the storage modulus in the shear deformation range of >1 was observed.The change in rheology is also visible in the phase lag values, for Pf-positive P. aeruginosa it increased above 60˚in the range <0.1 of tangential strain, while for Pf-positive P. aeruginosa + C. albicans values close to completely viscous liquid were observed (Fig 7D).For Pf-negative P. aeruginosa, the phase lag did not change significantly in the range <0.1, while >1 a significant increase was observed, especially for Pf-positive P. aeruginosa + S. aureus to a value close to 90˚at ~10 shear strain.The viscosity of the biofilms decreased over the entire range from below 0.005 Pa for low shear rates to below 0.002 Pa for high shear rates (S6D Fig).

Ceragenin CSA-13 in combination with DNase I and pASP alters mechanical properties by fluidizing mono-and dual-species biofilms
Although pASP alone did not change the rheological behavior of the biofilm, it had a greater effect in combination with CSA-13 than CSA-13 alone (Fig 7B and 7E; S6B and S6E Fig) .This combination reduced the storage modulus of biofilms (in terms of small <0.1 deformations in the amplitude sweep and frequency sweep), for Pf-negative P. aeruginosa + S. aureus by nearly 80%, for Pf-negative P. aeruginosa + C. albicans by over 75%, for Pf-positive P. aeruginosa + S. aureus and for Pf-positive P. aeruginosa + C. albicans by nearly 85%.For biofilms without bacteriophages, a rapid decrease in storage modulus was observed, accompanied by a rapid increase in phase lag in the range of >1 shear strain.The viscosity of these biofilms fell below 0.01 Pa over the entire range of shear rates (S6E Fig).
Significant rheological changes were also observed after treatment of biofilms with CSA-13 + Pf1 and are greater than when CSA-13 alone was used.In the range of small strains in the amplitude sweep and frequency sweep tests, a decrease in the storage modulus below 0.1 Pa was observed for all tested biofilms except for Pf-positive P. aeruginosa + C. albicans, for which the values were close to the control (untreated) (Fig 7C and 7F).For these biofilms, a sharp decrease in storage modulus and an accompanying increase in phase lag >0.1 tangent strain was also observed.The Pf-positive P. aeruginosa + C. albicans biofilm viscosity did not decrease significantly below the values obtained for the control, while for the remaining biofilms a decrease below 0.1 Pa was observed in the entire range of tangential deformations (S6E Fig).

Discussion
Mixed biofilm-associated infections, such as those observed in cystic fibrosis (CF) lung disease, require broad-spectrum and microbicidal antimicrobials that retain activity in polymer-rich environment [35][36][37][38].The viscoelasticity of CF biofilms is strongly influenced by species-specific factors, such as various P. aeruginosa exopolysaccharides (Psl, Pel, and alginate) and filamentous (Pf) bacteriophages [12,17].For instance, Psl promotes the growth of elastic biofilms with highly efficient cross-linking, whereas Pel and alginate favor the formation of viscoelastic and loose biofilms [12,39,40].Pel and Psl polymers also differently mediate P. aeruginosa interactions in mixed-species biofilm models, e.g., with S. aureus [39] which, along with species variable susceptibility patterns, promote the development of high tolerance to antimicrobial agents [41,42], and significantly impede the treatment of CF patients [35][36][37].Similarly, Pf bacteriophages elevate biofilm viscosity and create protective nanochambers enclosing bacterial cells [18,43].In addition, these ~2 μm long wire-shaped viruses interact with various biofilm polymers (DNA, actin, mucin, and glycosaminoglycans), resulting in a highly ordered liquid crystal formation promoting bacterial adhesion, desiccation survival, and antibiotic tolerance [44].The latter appears to be mediated via (i) low antibiotic penetration through lattice-like networks as a diffusion-limiting barrier, (ii) bacterial cell encapsulation in liquid crystalline droplets (nanochambers), and (iii) sequestration of cationic antibiotics, like aminoglycosides, by negatively charged virions [18,43,44].Recently, it was discovered that P. aeruginosa Pf1 and Pf4 phages act as an iron (Fe) chelators and inhibit C. albicans and Aspergillus fumigatus metabolism and biofilm formation via iron sequestration and denying this essential micronutrient to the fungi [45,46].This finding has potential practical implications since C. albicans is a prominent fungal pathogen in CF airways [8][9][10][11]47].Therefore, anti-biofilm therapies commonly involve various adjuvants that facilitate the action of antimicrobial agents, either by releasing the microbial cells from the protective matrix or by preventing their interactions with its components.Furthermore, novel antibacterial and antifungal compounds, such as essential oils derived from aromatic and medicinal plants, are continuously evaluated as a valuable natural alternative to conventional antibiotics [48][49][50][51].
Consistently, we evaluated the efficacy of the bactericidal and fungicidal ceragenin CSA-13 in combination with mucolytic agents-DNase I and pASP acid, against P. aeruginosa monoand bi-species (S. aureus and C. albicans) biofilms.To address the significance of P. aeruginosa filamentous (Pf) bacteriophages-a strain-specific biofilm viscoelastic trait-biofilms formed by Pf-positive and Pf-negative P. aeruginosa isolates were characterized by rheology and compared with the biofilms artificially supplemented with the exogenous Pf1 bacteriophage.
In general, Pf-positive P. aeruginosa mono-and dual-species biofilms had elevated viscosity and elasticity parameters when compared to Pf-negative ones (Figs 6A, 6E, 6J, 7C and 7F; S5E, S5J, S6C and S6F Figs).Pf-positive biofilm masses were less affected by CSA-13, either alone or in combination with DNase I, pASP acid, and exogenous bacteriophage Pf1.However, statistical significance was observed only for some concentrations of these compounds.
For instance, CSA-13 at a concentration of 5 μg/mL significantly decreased biofilm mass in the Pf-negative P. aeruginosa group but not at four times higher concentration (20 μg/mL).The total saturation of the biofilm matrix by this amount of CSA-13, and a massive reduction in biofilm mass in both groups, as with conventional antibiotics, is a plausible explanation for this observation [52].On the other hand, in mixed-species P. aeruginosa, S. aureus, and C. albicans biofilms, a significant impact of Pf phages was recorded only for CSA-13 at a dose of 20 μg/mL.This result might be related to the enhanced resistance of multi-species P. aeruginosa biofilms to conventional antibiotics, such as flucloxacillin, ciprofloxacin, and fluconazole, mediated by diffusion-limiting effects and/or the presence of matrix-associated degradative enzyme [3].DNase I improved the efficiency of CSA-13 at concentrations of 5 and 20 μg/mL but these differences were not statistically significant in Pf-positive group.
In contrast, the highest doses of both compounds significantly affected the biofilms of Pfnegative isolates (Fig 2H).This result might be related to enhanced biofilm penetration by CSA-13 due to eDNA degradation and, as a result, lower acidity of the matrix, as with other antimicrobial agents [2].At the same time, Pf phages can shield bacterial cells by enclosing them in nanochambers made of liquid crystalline droplets [43,44].The nanochambers are an example of a higher-order structure formed by filamentous phages due to their spontaneous self-assembly due to crowding or depletion-mediated attraction mechanisms, which are facilitated by a wide range of biopolymers (eDNA, alginate, or hyaluronan), and significantly impacted by the shape and size complementarity of the liquid crystals with rod-shaped bacterial cells [43,44].The range and intensity of depletion attraction are proportional to the size and concentration of biopolymers, thus, larger and/or more concentrated polymers favor liquid crystal assembly from lower Pf phage concentrations.Therefore, the degradation of eDNA by DNase I might introduce quantitative and qualitative alterations in the composition of biopolymers that contribute to the formation of liquid crystal nanochambers, e.g., via the altered ratio of DNA length or between DNA and other biopolymers [53] as well as by reducing cellcell adhesion [43,54], leading to increased bacterial survival.
The DNA concentration, the microbial cell morphology, and the number of microorganisms are essential determinants of biofilm mechanics [55,56].For instance, compression-stiffening and shear-softening features of biofilms can be recapitulated and modulated by modifying the DNA and microbial cell composition [56].A higher eDNA-to-cells ratio strengthens the biofilm's structure and enhances its viscoelastic moduli [56].Moreover, the level of compression-strengthening varies among microbial cell types and is more noticeable for P. aeruginosa than for S. aureus or C. albicans [56].Therefore, the presence of various cells in mixed biofilms may interfere with the mechanics of DNA, possibly by providing additional cross-links and restricting the space into which DNA can relocate throughout macroscopic deformation [56].Likewise, the stiffness of P. aeruginosa biofilms increases with the concentration of Pf1 bacteriophage [56].As a result, the viscoelastic properties of biofilms may involve mechanisms other than the interaction of cellular elements with the EPS matrix, as evidenced by the mechanical effects of cross-linking in the presence of bivalent ions and the interaction of biofilm components with Pf bacteriophages [56].
For instance, eDNA forms a filamentous network with a honeycomb-like structure and uniform thickness between bacterial cells [57], which may impair the assembly of Pf phages.eDNA is fundamental for the biofilm interactions with other biopolymers, including the major P. aeruginosa exopolysaccharides-a neutral Psl and cationic Pel, [58].For example, eDNA-Psl fibers act as a skeleton supporting bacterial adherence and growth and protecting biofilms against degradation by DNase I [53].However, Psl molecules can also be cross-linked by the protein CdrA, reinforcing the elastic network of the biofilm matrix [40].Likewise, Pel stabilizes the matrix by ionic interactions that bind eDNA with the biofilm [59].Furthermore, eDNA promotes the formation of amyloid fibers in bacterial biofilms, i.e., long and thin structures extending from the surface of the bacterial outer membrane, that sequester small metabolites, including essential for bacterial intercellular communication and biofilm formation quorum-sensing molecules [60].
In contrast, pASP, which like Pf1 is an anionic polyelectrolyte, alone at all concentrations (1-200 μg/mL) has negligible anti-pseudomonal biofilm activity or appears to slightly promote biofilm formation, as reflected by generally minor rheological differences compared to untreated biofilms.Therefore, in terms of biofilm mass, the effect of pASP on P. aeruginosa biofilm formation is comparable to that mediated by exogenous bacteriophage Pf1 (Fig 2B and  2C).The above is reflected in our previous results, which clearly indicate that the activity of pASP depends on its polycationic charge [61].
Subsequently, the combination of CSA-13 at the dose of 20 μg/mL, even with the highest amounts of pASP acid, i.e., 100 and 200 μg/mL, was not superior over CSA-13 alone in the reduction of biofilm mass in Pf-positive P. aeruginosa isolates.In contrast, biofilms of Pf-negative P. aeruginosa strains were significantly more affected by both combinations.It remains to be elucidated whether this difference is related to the depletion attraction phenomenon [44], where more concentrated pASP favors the assembly of Pf phages into liquid crystals.Alternatively, since specific D-and L-amino acid isoforms, including aspartic acid, have anti-biofilm activity [38,62,63], it might be assumed that its release via degradation polyaspartic acid may inhibit biofilm formation in Pf-negative P. aeruginosa isolates.
Since pASP acid is an anionic polyelectrolytes, it might exhibit the same behavior as natural biopolymers present in P. aeruginosa biofilms, e.g., interacting via acidic (carboxyl) groups with cationic biopolymers [58], and due to the high water absorbency, pASP may promote the formation of highly hydrated biofilms.Furthermore, anionic polyelectrolytes undergo chain conformation changes from globular to a coil in the presence of electrolytes, which compensate for the repulsive Coulomb forces between anionic carboxyl groups responsible for the elongation of polymer chains, resulting at higher ion concentrations in an extensive agglomeration of the polymer and growing particle size [58].
The cationic CSA-13 might interfere with this process in a similar way to the cationic histone protein H2A disturbs interactions between alginate and calcium, leading to increased deformability of an alginate-calcium gel and reflected by decreased storage modulus G' and loss modulus G", but increased stress under strain [58,64].Indeed, a rapid decrease in storage modulus and increase in phase lag was observed in Pf-negative P. aeruginosa biofilms treated with the CSA-13/ pASP acid combination (Fig 7B and 7E; S6B and S6E Fig) .Like DNase I, ASP oligomers can decrease the elasticity of CF sputum or neutrophil-induced P. aeruginosa biofilms; however, acting via dissolving actin and DNA bundles by sequestration of cationic histone linking electrostatically these negatively charged biopolymers [5,16].
Our earlier research demonstrated a substantial reduction of the bactericidal action of natural peptide LL-37 in the presence of exogenous bacteriophage Pf1 as well as by Pf-positive P. aeruginosa LES strain without impairing ceragenins' activity [24].Here we extend this observation over additional P. aeruginosa isolates expressing Pf belonging to distinct evolutionary lineages and characterized by different structural/morphogenesis properties [21].The presence of Pf phages promotes biofilm formation (Fig 2C ) and alters biofilm rheological properties toward enhanced viscosity and elasticity (Figs 6E, 6J, 7C and 7F; S5E, S5J, S6C and S6F Figs).Therefore, targeting P. aeruginosa Pf phages might provide an alternative therapeutic approach for CF patients.Moreover, according to our best knowledge, this is the first study comparing the activity of CSA-13 in combination with DNase I and pASP in treating CF-associated bispecies biofilms.
In conclusion, this study confirms and expands the findings of our previous studies on the impact of polyelectrolytes, such as DNA and P. aeruginosa filamentous (Pf) bacteriophages, on rheological properties of biofilms through the investigation of antibiofilm activity of ceragenins (CSAs) and mucolytic agents-DNase I and pASP [56,65].CSA-13 is a potent microbicidal and biofilm fluidizing agent, especially when supported by DNase I or pASP acid, and appears to be a good candidate for further studies in the treatment of biofilms produced by P. aeruginosa strains, regardless of their Pf-profiles, and other CF-associated bacterial and fungal pathogens.Nevertheless, for a more reliable assessment of ceragenin antibiofilm efficacy, experiments must be performed under conditions mimicking CF lungs, such as artificial sputum media (ASM), synthetic cystic fibrosis media (SCFM), or CF sputum samples.Accordingly, we recently reported that ceragenins preserve bactericidal activity in the environment mimicking CF sputum irrespective of its ionic strength [66].Furthermore, the effect of the microbial cell morphology [56] and Pf bacteriophages representing various phylogenetic lineages [21] on the viscoelastic properties of biofilms should be addressed in further studies.

Fig 1 .Fig 2 .
Fig 1. (A) Minimal inhibitory concentrations and minimal bactericidal concentrations (MIC/MBC; μg/mL) of CSA-13, CSA-44, and CSA-131 against five Pf-negative P. aeruginosa strains (black bars) and five Pf-positive P. aeruginosa strains (blue bars).(B) Inhibitory concentration at 50%-level (IC50) was recorded for tested ceragenins (CSA-13, CSA-44, and CSA-131) against five Pf-negative P. aeruginosa strains (black bars) and Pf-positive P. aeruginosa strains blue bars).The IC50 values were calculated by interpolation of the dose-response curve when biofilm formation in the aureus and C. albicans strains (Fig 4A and S2A Fig).Furthermore, CSA-13, in combination with DNAse I or pASP acid, significantly decreased only the biofilm mass produced by Pf-negative P. aeruginosa with C. albicans (Fig 4E, 4F, 4H and 4J, and S2B and S2C Fig).CSA-13 activity was retained in dual-biofilms supplemented with the exogenous Pf1 (Fig 4G-4J and S2D Fig).Viability of mono-and dual-species biofilms, assessed by CFU counting, (Fig 5A-5G and S3A-S3G Fig) revealed that DNase I (Fig 5B and S3B Fig) and pASP (Fig 5C and S3C Fig) are more effective against S. aureus and C. albicans than against P. aeruginosa.Viscoelasticity of biofilms depends on P. aeruginosa Pf-profileUntreated mono-and dual-biofilms developed by Pf-positive and Pf-negative P. aeruginosa co-cultured with S. aureus/C.albicans strains differ in rheological properties, as shown in S4A-S4D Fig as well as Fig 6A and 6F, respectively.The co-culture of Pf-positive P. aeruginosa and S. aureus or C. albicans results in biofilms with significantly higher rigidity compared to co-cultures with Pf-negative P. aeruginosa (Fig 6A).Pf-positive P. aeruginosa biofilms, especially those co-developed with S. aureus, have a higher storage modulus and a smaller decrease in the amplitude sweep test.For Pf-negative P. aeruginosa + S. aureus, G' values decreases from 0.47 Pa to 0.08 Pa, for Pf-negative P. aeruginosa + C. albicans G' values decreases from 0.29 Pa to 0.05 Pa, for Pf-positive P. aeruginosa + S. aureus G' values decreases from 0.56 Pa to 0.32 Pa, and for Pf-positive P. aeruginosa + C. albicans G' values decreases from 0.38 Pa to 0.12 Pa (Fig 6A)

Fig 4 .
Fig 4. The relative mass of polymicrobial biofilms formed by Pf-negative and Pf-positive P. aeruginosa isolates with S. aureus Xen30 (SA, red bars), and C. albicans 1408 (CA, grey bars) strains in dual-species mixtures.Recorded values were compared to the anti-biofilm activity of CSA-13 alone (A).Biofilm mass was measured in the presence of DNase I (B), pASP (C), and Pf1 bacteriophage (D) at concentrations from 0.5 to 5 μg/mL, 1 to 200 μg/mL and 0.01 to 0.5 mg/mL, respectively and co-administrated with CSA-13 at doses of 5 (E-G) or 20 μg/mL (H-J).Results are presented as mean ± SD of three repetitions.* indicates statistical significance when compared to dual-species mixtures of biofilm with Pf-negative P. aeruginosa isolates.https://doi.org/10.1371/journal.pone.0298112.g004

Fig 5 .
Fig 5. Viability of polymicrobial biofilms formed by Pf-negative and Pf-positive P. aeruginosa isolates, S. aureus (SA), and C. albicans (CA) strains in dual-species mixtures.Recorded values were compared to the anti-biofilm activity of CSA-13 alone (A).Biofilm viability was measured in the presence of DNase I (B), pASP (C) and Pf1 bacteriophage (D) at concentrations of 5 μg/mL, 10 μg/mL, and 0.1 mg/mL, respectively and co-administrated with CSA-13 at dose of 20 μg/mL (E-G).Green bars indicate percentage of bacteria grown.Results are presented as mean ± SD of three repetitions.https://doi.org/10.1371/journal.pone.0298112.g005 . The phase shift over the entire range increased above 45˚for all samples, indicating a transition of the biofilm to a more fluid-like consistency (Fig 6G).Similar observations were yielded by frequency sweep tests, wherein the range of 0.1-10 Hz the storage modulus values decreased more than 4 times for Pf-negative P. aeruginosa + S. aureus, more than twice for Pf-negative P. aeruginosa + C. albicans, and Pf-positive P. aeruginosa + C. albicans, and nearly 3 times for Pf-positive P. aeruginosa + S. aureus (S5B Fig).The viscosity of CSA-treated biofilms also decreased, especially evident for Pf-positive P. aeruginosa + S. aureus and Pf-positive

Fig 6 .
Fig 6.Rheological measurements of polymicrobial biofilms formed by Pf-negative and Pf-positive P. aeruginosa isolates with S. aureus, and C. albicans strains in dual-species mixtures upon treatment with single compound.Shear storage modulus as a function of shear strain amplitude (A-E) and the phase shift as a function of shear strain amplitude (F-J).Rheological properties of biofilms were measured in the presence of CSA-13 (B, G), DNase I (C, H) pASP (D, I) and Pf1 bacteriophage (E, J) at concentrations of 20 μg/mL, 5 μg/mL, 10 μg/mL and 0.1 mg/mL, respectively.Recorded values were compared to the control-without treatment (A, F). https://doi.org/10.1371/journal.pone.0298112.g006 Fig 6C).Biofilm viscosity also decreased, for Pf-negative P. aeruginosa + S. aureus it decreased by almost 70%, for Pfnegative P. aeruginosa + C. albicans it decreased by about 30%, for Pf-positive P. aeruginosa + S. aureus by over 70%, and for Pf-positive P. aeruginosa + C. albicans by nearly 50% (S5H Fig).The addition of pASP slightly affected the rheological behavior of the biofilm.The only significant differences were observed in the overall decrease of several percent in the phase shift of Pf-positive P. aeruginosa + C. albicans and the more viscous behavior of Pf-negative P. aeruginosa + S. aureus in the range above 10 shear strain (Fig 6D, S5I Fig).The addition of Pf1 had a greater impact on the biofilm mechanics, increasing the storage modulus by several percent and significantly shifting the phase lag of C. albicans biofilms (by about 15˚for Pf-negative P. aeruginosa + C. albicans and by about 11˚for Pf-positive P. aeruginosa + C. albicans) towards more elastic behavior.

Fig 7
Fig 7 shows the rheological behavior of biofilms of the same strain configurations co-treated with CSA-13 and DNase I, pASP, or Pf1.The addition of CSA-13 and DNase I significantly reduced the storage modulus of biofilms for Pf-negative P. aeruginosa + S. aureus by over 90%,

Fig 7 .
Fig 7. Rheological measurements of polymicrobial biofilms formed by Pf-negative and Pf-positive P. aeruginosa isolates with S. aureus, and C. albicans strains in dual-species mixtures upon treatment with co-administrated agents.Shear storage modulus as a function of shear strain amplitude (A-C) and the phase shift as a function of shear strain amplitude (D-F).Rheological properties of biofilms were measured with CSA-13 at dose of 20 μg/mL in the coadministrated with DNase I (A, D) pASP (B, E) and Pf1 bacteriophage (C, F) at concentrations of 5 μg/mL, 10 μg/mL and 0.1 mg/mL, respectively.https://doi.org/10.1371/journal.pone.0298112.g007